The p53 gene is one of the most studied and well-known genes. p53 plays a key role in cellular stress response mechanisms by converting a variety of different stimuli, for example, DNA damage, deregulation of transcription or replication, and oncogene transformation, into cell growth arrest or apoptosis (Kastan et al., Cancer Res 1991; 51:6304-6311; Kastan et al., Cell 1992; 71:587-597; Vogelstein et al., Nature 2000; 408:307-310; Vousden et al., Nat Rev Cancer 2002; 2:594-604; Giaccia and Kastan, Genes & Development 1998; 12:2973-2983.
The p53 protein is active as a homo-tetramer and exerts its tumor suppressor function mainly as a transcription factor that affects G1 and G2 cell cycle arrest and/or apoptosis (see, e.g., Donehower and Bradley, Biochim Biophys Acta., 1993, 1155(2):181-205; Haffner and Oren, Curr. Opin. Genet. Dev., 1995, 5(1):84-90; Gottlieb and Oren, Biochim. Biophys. Acta., 1996, 1287(2-3):77-102; Ko and Prives, Genes Dev., 1996, 10(9):1054-72; Hansen and Oren, Curr. Opin. Genet. Dev., 1997, 7(1):46-51; Levine, Cell, 1997, 88(3):323-31). The p53-mediated G1 arrest is its best characterized activity and involves transcriptional activation of the downstream gene p21 WAF1/CIP1/SDI1 (Haffner and Oren, Curr. Opin. Genet. Dev., 1995, 5(1):84-90; Gottlieb and Oren, Biochim. Biophys. Acta., 1996, 1287(2-3):77-102; Ko and Prives, Genes Dev., 1996, 10(9):1054-72; Hansen and Oren, Curr. Opin. Genet. Dev., 1997, 7(1):46-51; Levine, Cell, 1997, 88(3):323-31). Other downstream effector genes for p53-mediated G1 arrest may exist, since p21−/− mouse embryonic fibroblasts do not show complete abrogation of GI arrest after DNA damage (Brugarolas et al., Nature, 1995, 377(6549):552-7; Deng et al., Cell, 1995, 82(4):675-84). The G2/M effects of p53 involve, at least in part, induction of 14-3-3σ (Hermeking et al., Mol. Cell, 1997, 1(1):3-11).
The mechanisms for apoptosis induction and their relative importance remain less clear at present. In certain settings p53 clearly induces pro-apoptotic genes. These include BAX and Fas/APO1 (Miyashita and Reed, Cell, 1995, 80(2):293-9; Owen-Schaub et al., Mol. Cell. Biol., 1995, 15(6):3032-40) neither of which, however, is an absolute requirement for p53-induced apoptosis (Fuchs et al., Cancer Res., 1997, 57(13):2550-4). Recently, many more genes have been identified that are induced directly or indirectly during p53-mediated apoptosis (Polyak et al., Nature, 1997, 389(6648):300-5), but the essential genes for p53-induced apoptosis still have to be determined. Transcriptional repression of anti-apoptotic genes, such as bcl-2, may play a role (Haldar et al., Cancer Res., 1994, 54(8):2095-7; Miyashita et al., Oncogene, 1994, 9(6):1799-805) and other non-transcriptional mechanisms may be important as well (Caelles et al., Nature, 1994, 370(6486):220-3; Haupt et al., Nature 1997; 387:296-299).
Several upstream signals activate p53. These include DNA damage, hypoxia and critically low ribonucleoside triphosphate pools (Kastan et al., Cancer Res. 1991; 51:6304-6311; Graeber et al., Nature, 1996, 379(6560):88-91; Linke et al., Genes Dev., 1996, 10(8):934-47). Once activated, p53 induces either cell cycle arrest or apoptosis, depending on several factors such as the amount of DNA damage, cell type and cellular milieu, e.g., presence or absence of growth factors (Donehower and Bradley, Biochim Biophys Acta., 1993, 1155(2):181-205; Haffner and Oren, Curr. Opin. Genet. Dev., 1995, 5(1):84-90; Gottlieb and Oren, Biochim. Biophys. Acta., 1996, 1287(2-3):77-102; Ko and Prives, Genes Dev., 1996, 10(9):1054-72; Hansen and Oren, Curr. Opin. Genet. Dev., 1997, 7(1):46-51; Levine, Cell, 1997, 88(3):323-31); Giaccia and Kastan, Genes & Development 1998; 12:2973-2983.
Cancer cells show decreased fidelity in replicating their DNA, often resulting in DNA damage, and tumor masses have inadequate neovascularization leading to ribonucleoside triphosphate or oxygen deprivation, all upstream signals that activate p53. In view of p53's capability to induce cell cycle arrest or apoptosis under these conditions it is not surprising that absent or significantly reduced activity of the tumor suppressor protein p53 is a characteristic of more than half of all human cancers (Harris and Hollstein, N. Engl J. Med., 1993, 329(18):1318-27; Greenblatt et al., Cancer Res., 1994, 54(18):4855-78). In the majority of cancers, p53 inactivation is caused by missense mutations in one p53 allele, often with concomitant loss-of-heterozygosity (Michalovitz et al., J. Cell. Biochem., 1991, 45(1):22-9; Vogelstein and Kinzler, Cell, 1992, 70(4):523-6; Donehower and Bradley, Biochim. Biophys. Acta., 1993, 1155(2):181-205; Levine, Cell, 1997, 88(3):323-31). These mutations affect almost exclusively the core DNA-binding domain of p53 that is responsible for making contacts with p53 DNA-binding sites, while mutations in the N-terminal transactivation domain or the C-terminal tetramerization domain are extremely rare (Beroud and Soussi, Nucleic Acids Res., 1998, 26(1):200-4; Cariello et al., Nucleic Acids Res., 1998, 26(1):198-9; Hainaut et al., P., Nucleic Acids Res. 1998; 26:205-213).
Contrary to wild-type p53, p53 cancer mutants have a long half-life and accumulate to high levels in cancer cells (Donehower and Bradley, Biochim Biophys Acta., 1993, 1155(2):181-205; Lowe, Curr. Opin. Oncol., 1995, 7(6):547-53). This may be explained by their inability to activate the mdm-2 gene (Lane and Hall, Trends Biochem. Sci., 1997, 22(10):372-4), since mdm-2 induces degradation of p53 via the ubiquitin pathway as part of a negative feedback loop (Haupt et al., Nature 1997; 387:296-299; Kubbutat et al., Nature 1997; 387:299-303). The unusually high frequency of p53 missense mutations in human cancers (as opposed to mutations resulting in truncated proteins) is explained by their dominant-negative effect that depends on the intact C-terminal tetramerization domain. The C-terminus allows p53 cancer mutants to form hetero-tetramers with wild-type p53 (Milner and Medcalf, Cell, 1991, 65(5):765-74), thus reducing, or even abrogating, the activity of wild-type p53 protein (Michalovitz et al., J. Cell. Biochem., 1991, 45(1):22-9; Vogelstein and Kinzler, Cell, 1992, 70(4):523-6; Ko and Prives, Genes Dev., 1996, 10(9):1054-72). In addition, there is evidence that at least some of the same missense mutations may confer a gain-of-function (Gottlieb and Oren, Biochim. Biophys. Acta., 1996, 1287(2-3):77-102; Ko and Prives, Genes Dev., 1996, 10(9):1054-72; Levine, Cell, 1997, 88(3):323-31).
p53 imparts sensitivity to normal tissues subjected to genotoxic stress. For example, p53-mediated apoptosis causes side effects of radiation therapy and chemotherapy such as severe damage to the lymphoid and hematopoietic system and intestinal epithelia, which limit the effectiveness of these therapies. [Gudkov and Komarova, Nat. Rev. Cancer, 2003, 3:117-129; Westphal et al., Cancer Research, 1998]. Other side effects, like hair loss, also are p53 mediated and further detract from cancer therapies (Botchkarev et al., Cancer Res., 2000, 60:5002-5006).
In summary, p53 has a dual role in cancer therapy. On one hand, p53 acts as a tumor suppressor by mediating apoptosis and growth arrest in response to a variety of stresses and controlling cellular senescence. On the other hand, p53 is responsible for severe damage to normal tissues during cancer therapies.
The damage caused by p53 to normal tissues makes p53 a potential target for therapeutic suppression. Since more than 50% of human tumors lack functional p53, suppression of p53 would not affect the efficacy of a treatment for such tumors, and would protect normal p53-containing tissues (Komarova et al., Seminars in Cancer Biology 1998; 8(5):389-400). It has been also recognized that therapeutic p53 inhibition should be reversible as long-term p53 inactivation can significantly increase the risk of cancer. Komarov et al. (Science, 1999; 285:1733-1737) developed a cell-based screen aimed at identifying compounds capable of inhibiting p53-mediated apoptosis from a library of 10 000 synthetic chemicals. In this screen, they have identified a stable water-soluble p53-inhibitor pifithrin-α (PFT-α), which suppressed p53-dependent apoptosis and protected mice from the lethal genotoxic stress associated with cancer treatment without promoting the formation of tumors. See also, e.g., U.S. Pat. Nos. 7,012,087; 6,593,353 and 6,420,136. PFT-α does not block p53 induction and the mechanism by which it functions downstream of p53 has not been elucidated yet. Further, it has been recently shown that PFT-α protects cells from DNA damage-induced apoptosis also by a p53-independent mechanism (Sohn et al., Cell Death and Differentiation (2009), 1-10). Such complexity of the mechanism of action makes PFT-α less attractive as a p53 inhibitory therapeutic, since all possible side effects cannot be easily assessed. Thus, novel compounds are needed which would selectively and reversibly inhibit p53 activity in normal tissues during treatment of p53-deficient tumors, and thereby protect normal tissues.
The adverse effects of p53 activity on an organism are not limited to cancer or cancer therapies. p53 is activated as a consequence of a variety of stresses associated with injuries (e.g., burns), naturally occurring diseases (e.g., fever, and conditions of local hypoxia associated with a blocked blood supply, stroke, and ischemia) and cell aging (e.g., senescence of fibroblasts). p53 inhibition, therefore, also can be therapeutically effective, for example, in reducing or eliminating p53-dependent neuronal death in the central nervous system (e.g., after brain and spinal cord injury), reducing or eliminating neuronal damage during seizures, suppressing tissue aging, or preservation of tissues and organs prior to transplantation.
p53 regulation has also been shown to affect the pathogenesis of neurodegenerative diseases. For example, as shown by Bae et al. (Neuron 2005; 47:29-41), (i) p53 levels are increased in the brains of mutant huntingtin protein (mHtt) transgenic mice (mHtt-Tg) and Huntington's Disease (HD) patients and (ii) upregulation of p53 transcriptional activity and nuclear p53 levels by mHtt leads to mitochondrial depolarization and cytotoxicity in neuronal cell cultures, revealing a role for p53 regulation in the development of HD. Reduction or elimination of p53 suppresses this neurodegenerative effect. Thus, p53 regulation can be beneficial for amelioration of HD and other neurodegenerative diseases.
p53 has a short half-life, and, accordingly, is continuously synthesized and degraded in the cell. However, when a cell is subjected to stress (e.g., (a) DNA damage, such as damage caused by ionizing irradiation (IR) or UV (ultraviolet) radiation, cell mutations, chemotherapy, and radiation therapy; (b) hyperthermia; and (c) deregulation of microtubules caused by some chemotherapeutic drugs, e.g., treatment using taxol or Vinca alkaloids), the intracellular levels of functional p53 protein increase (Canman et al., Oncogene 1998; 16:957-966; Canman et al., Genes & Dev. 1995; 9:600-611; Kuerbitz et al., Proc Natl Acad Sci 1992; 89:7491-7495). The increases in p53 protein levels are dependent on the ATM protein kinase after ionizing irradiation (IR) (Kastan et al., Cell 1992; 71:587-597) and on the ATR protein kinase after UV irradiation and many other types of cellular stress (Tibbetts et al., Genes & Development 1999; 13:152-157; Hammond et al., Mol Cell Biol. 2002; 22:1834-1843; Wright et al., Pro Natl Acad Sci U.S.A. 1998; 95:7445-7450).
There is a measurable increase in the half-life of p53 protein after DNA damage (Maltzman et al., Molec and Cell Biol 1984; 4(9):1689-1694; Price et al., Oncogene 1993; 8:3055-3062; Maki et al., Mol. Cell Biol. 1997; 17:355-363) and the increases in cellular p53 protein levels have largely been attributed to this change in half-life. p53 protein is normally a very short-lived cellular protein with rapid proteosomal degradation in unperturbed cells. The HDM2 protein (MDM2 in mice) directly binds to p53 protein (Momand et al., Cell 1992; 69:1237-1245; Oliner et al., Nature 1993; 362:857-860) and functions as an E3 ubiquitin ligase to facilitate the degradation of p53 (Fang et al., S., J Biol Chem 2000; 275:8945-8951; Honda et al., FEBS Letters 1997; 420:25-27; Haupt et al., Nature 1997; 387:296-299; Kubbutat et al., Nature 1997; 387:299-303). Post-translational modifications of HDM2 and p53 after DNA damage appear to inhibit the ability of HDM2 to bind to p53 (Mayo et al., Cancer Research 1997; 57:5013-5016; Khosravi et al., PNAS 1999; 96:14973-14977; Maya et al., Genes & Development 2001; 15:1067-1077; Shieh et al., Cell 1997; 91:325-334; Ashcroft et al., Molecular & Cellular Biology 1999; 19:1751-1758), thus decreasing the proteasomal degradation of p53 protein and increasing cellular levels of the protein. Similarly, induction of the ARF tumor suppressor by oncogenes and other cellular signals leads to increases in p53 protein levels by ARF protein binding to HDM2 and inhibiting HDM2-mediated degradation of p53 (Palmero et al., Nature 1999; 395:127; Kamijo et al., Proc. Natl. Acad. Sci. U.S.A 1998; 95:8292-8297; Sherr et al., Curr. Opin. Genet. Dev. 2000; 10:94-99; Pomerantz et al., Cell 1998; 92:713-723; Stott et al., EMBO J. 1998; 17:5001-5014). Thus, cells with overexpressed HDM2 or inactive ARF are similar to cells containing mutated p53 genes in that normal p53 regulation is lacking.
It has been recently established that translational regulation also contributes to p53 induction after DNA damage (Takagi et al., Cell, 2005, 123(1): 49-63). In the initial reports of p53 induction after ionizing irradiation, the protein synthesis inhibitor cycloheximide was shown to block p53 induction and marked increases in labeling of p53 protein with [35S]-methionine were noted early after treatment (Kastan et al., Cancer Res 1991; 51:6304-6311; Kastan et al., Cell 1992; 71:587-597). Subsequently, a translation suppressor element was reported in the 3′-UTR of the p53 mRNA (Fu et al., EMBO J. 1997; 16:4117-4125; Fu et al., Oncogene 1999; 18:6419-6424; Fu et al., EMBO J. 1996; 15:4392-4401) and a stem loop structure was predicted in the 5′-UTR of the murine p53 gene (Mosner et al., EMBO J. 1995; 14:4442-4449). Interestingly, p53 was suggested to negatively regulate its own translation by direct binding of p53 protein to this 5′-UTR stem loop structure (Mosner et al., EMBO J. 1995; 14:4442-4449). Two other proteins have also been reported to modulate p53 translation: thymidylate synthase suppresses p53 translation by binding to the coding sequence of p53 mRNA (Chu et al., Mol. Cell Biol. 1999; 19:1582-1594; Ju et al., Proc. Natl. Acad. Sci. U.S.A 1999; 96:3769-3774) and HuR (Hu antigen R) enhances the translation efficiency of p53 after ultraviolet irradiation by binding to an AU-rich sequence at the 3′-UTR of p53 mRNA (Mazan-Mamczarz et al., Proc. Natl. Acad. Sci. U.S.A 2003; 100:8354-8359).
Screens for proteins that specifically bind to a 5′-UTR of p53 mRNA conducted by the present inventors have identified three proteins, Ribosomal Protein L26 (RPL26) (e.g., human RPL26 protein having GenBank Accession No. NP—000978) (SEQ ID NO: 49), nucleolin (“NCL”) (e.g., human nucleolin protein having GenBank Accession No. NP—005372) (SEQ ID NO: 51), and p53 protein itself (e.g., human p53 protein having GenBank Accession No. NP—000537) (SEQ ID NO: 54), that bind to the 5′UTR of p53 both in vitro and in cells (see PCT Publication No. WO 2007/041213 and Takagi et al., Cell, 2005, 123(1): 49-63). Manipulations of RPL26 and nucleolin demonstrated that they modulate p53 protein levels and affect p53 induction after DNA damage. Increased levels of RPL26 enhance both basal and DNA damage-induced translation of p53 mRNA in vitro and in cells and enhance cellular functions dependent on p53, such as cell cycle arrest and apoptosis. The effects of RPL26 on p53 translation require the presence of the 5′-UTR. Reduction of RPL26 levels by siRNA inhibit these p53-dependent responses, thus demonstrating a role for endogenous RPL26 in DNA damage responses. Nucleolin has the opposite effects on p53, with overexpression reducing basal and DNA damage-induced translation and inhibition of nucleolin enhancing translation. Taken together, RPL26 and nucleolin appear to compete with each other to regulate p53 synthesis through binding to a 5′-UTR of p53 mRNA.
It has been recently reported (Ofir-Rosenfeld et al., Mol. Cell., 2008, 32: 180-189) that translational regulation of p53 also involves protein Mdm2 As noted above, Mdm2 regulates p53 protein by promoting its proteasome-mediated degradation, and Mdm2 and p53 engage in an autoregulatory feedback loop that maintains low p53 activity in nonstressed cells. As shown by Ofir-Rosenfeld et al., Mdm2 also regulates p53 levels by targeting RPL26. Mdm2 binds RPL26 and drives its polyubiquitylation and proteasomal degradation. In addition, the binding of Mdm2 to RPL26 attenuates the association of RPL26 with p53 mRNA and represses RPL26-mediated augmentation of p53 protein synthesis. It is hypothesized that under nonstressed conditions, both mechanisms help maintain low cellular p53 levels by constitutively tuning down p53 translation, while, in response to genotoxic stress, the inhibitory effect of Mdm2 on RPL26 is attenuated, enabling a rapid increase in p53 synthesis.
Currently, there are two recognized forms of eukaryotic translation, cap-dependent translation and cap-independent translation (Hellen and Sarnow, 2001, Genes Dev. 15: 1593-1612). The recognition of the 7-methylguanosine cap located at the 5′-end of eukaryotic mRNAs by the eukaryotic initiation factor eIF4E, which is part of a greater initiation complex eIF4F, is a crucial step of cap-dependent protein translation (Gingras et al., 1999, Annu. Rev. Biochem. Allied Res. 68: 913-963). During cellular stress, such as heat shock (Vries et al., 1997, J. Biol. Chem. 272: 32779-32784) or hypoxia (Tinton and Buc-Calderon, 1999, FEBS Lett. 446: 55-59), 4E-BP1 is dephosphorylated and cap-dependent protein translation is impaired. In these instances, cap-independent protein translation initiation, which does not require the presence of the 7-methylguanosine cap or its binding factor, eIF4E, is used to synthesize the needed proteins (Vagner et al., 2001, EMBO J., 2: 893-898). Cap-independent translation is usually mediated by a complex structural element at the 5′-UTR of the mRNA called an internal ribosome entry site (IRES). With the help of eIF4G and other translation initiation factors, IRESs are capable of recruiting the 40S ribosomal subunit and initiating translation without the need for the initiation factor eIF4E. Recent findings show that p53 can be translated in a cap-independent manner, e.g., in response to treatment with the DNA-damaging agent etoposide (Yang et al., 2006, Oncogene 25: 4613-4619; Ray et al., 2006, EMBO Rep. 7: 404-410). Deletion analysis demonstrated that most of the p53 IRES activity is contained within the first 70 nucleotides of the p53 5′-UTR (Yang et al., 2006, Oncogene 25: 4613-4619).
Taken together, both the 5′- and 3′-untranslated regions (UTR) of the p53 mRNA appear to be sites of p53 translational regulation in response to stress through various trans-acting factors.